Therapeutic Effect of Bee Venom and Melittin on Skin Infection Caused by Streptococcus pyogenes

Abstract

Streptococcus pyogenes (S. pyogenes) bacteria cause almost all primary skin infections in humans. Bee venom (BV) and melittin (Mel) have multiple effects, including antibacterial and anti-inflammatory activities. This study aims to demonstrate their effects on bacterial mouse skin infection using S. pyogenes. The dorsal skin was tape-stripped, then S. pyogenes was topically applied. BV or Mel were topically applied to the lesion. The tissues were stained with hematoxylin and eosin, while immunohistochemical staining was performed with anti-neutrophil. S. pyogenes-infected skin revealed increased epidermal and dermal layers, but it was reduced in the BV and Mel groups. Finding increased neutrophils in the mice infected with S. pyogenes, but the BV and Mel mice showed decreased expression. These results suggest that BV and Mel treatments could reduce the inflammatory reactions and help improve lesions induced by S. pyogenes skin infection. This study provides additional assessment of the potential therapeutic effects of BV and Mel in managing skin infection caused by S. pyogenes, further suggesting that it could be a candidate for developing novel treatment alternative for streptococcal skin infections.

Keywords: Streptococcus pyogenes; bee venom; melittin; skin infection.

Figure 1

Skin lesions after S. pyogenes infection. Mice were superficially infected with S. pyogenes (1 × 10⁸ CFU/50 μL) and subsequently treated with BV or Mel, with the first application 48 h after infection and the second and final application 6 h later. At 18 h after the last treatment, the representative images of the back were photographed. Lesions appeared on the skin of all infected mice with a vesicle on an easily ruptured erythematous base, resulting in superficial ulceration covered with purulent discharge that then dried as an adherent yellow crust. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL BV cream (BV1, BV100, and BV500); S. pyogenes infection with 1, 100, and 500 μg/mL Mel cream (Mel1, Mel100, and Mel500).

Figure 2

Effect of BV in S. pyogenes skin infection models. At 18 h after the last topical application, biopsy specimens were taken and immediately fixed in 10% formalin, embedded in paraffin, and then stained with hematoxylin and eosin (H&E). (a) An increase in skin thickness and epidermal hyperplasia surrounding the infection site of S. pyogenes was observed by H&E staining. (b) Numerical value of epidermis thickness. (c) Numerical value of dermis thickness. The graphs are the statistical analysis for each staining. The results are expressed as mean ± SEM of three independent determinations. * p < 0.05 compared with the NC group; † p < 0.05 compared with the IN group. Magnification ×200, scale bar 50 μm. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL BV cream (BV1, BV100, and BV500).

Figure 3

Effect of Mel in S. pyogenes skin infection models. At 18 h after the last topical application, biopsy specimens were taken and immediately fixed in 10% formalin, embedded in paraffin, and then stained with hematoxylin and eosin (H&E). (a) An increase in skin thickness and epidermal hyperplasia surrounding the infection site of S. pyogenes was observed by H&E staining. (b) Numerical value of epidermis thickness. (c) Numerical value of dermis thickness. The graphs are the statistical analysis for each staining. The results are expressed as mean ± SEM of three independent determinations. * p < 0.05 compared with the NC group; † p < 0.05 compared with the IN group. Magnification ×200, scale bar 50 μm. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL Mel cream (Mel1, Mel100, and Mel500).

Figure 4

Effects of BV on neutrophil expression in S. pyogenes skin infection. (a) The representative immunohistochemical analysis images show that treatment with BV suppressed neutrophil expression in S. pyogenes-infected skin. (b) The graph shows the percentage of neutrophil-positive area. (c) Western-blot analysis shows the protein expressions of neutrophil in the skin tissues of each group. The results are expressed as means ± SEM. * p < 0.05 compared with the NC group; † p < 0.05 compared with the IN group. Magnification ×400, scale bar 40 μm. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL BV cream (BV1, BV100, and BV500).

Figure 5

Effects of Mel on neutrophil expression in S. pyogenes skin infection. (a) The representative immunohistochemical analysis images show that treatment with Mel suppressed neutrophil expression in S. pyogenes-infected skin. (b) The graph shows the percentage of neutrophil-positive area. (c) Western-blot analysis shows the protein expressions of neutrophil in the skin tissues of each group. The results are expressed as means ± SEM. * p < 0.05 compared with the NC group; † p < 0.05 compared with the IN group. Magnification ×400, scale bar 40 μm. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL Mel cream (Mel1, Mel100, and Mel500).

Figure 6

Effects of BV and Mel on the expression of IL-17A in S. pyogenes skin infection. (a,c) Immunochemical staining was performed to confirm the expression of IL-17A. (b,d) The graphs show the percentage of IL-17A-positive area. * p < 0.05 compared with the NC group; † p < 0.05 compared with the IN group. Magnification ×400, scale bar 40 μm. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL BV cream (BV1, BV100, and BV500); S. pyogenes infection with 1, 100, and 500 μg/mL Mel cream (Mel1, Mel100, and Mel500).

Figure 7

Effects of BV and Mel on the expression of pro-inflammatory cytokines. IL-1β and TNF-α were analyzed by Western-blot, and GAPDH was used to confirm equal loading of all protein samples. BV and Mel inhibit the expression of pro-inflammatory cytokines in S. pyogenes-infected skin tissues. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL BV cream (BV1, BV100, and BV500); S. pyogenes infection with 1, 100, and 500 μg/mL Mel cream (Mel1, Mel100, and Mel500).

Figure 8

Effects of BV and Mel on filaggrin expression in S. pyogenes–infected mice. (a) Effects of BV on skin barrier function. (b) Effects of Mel on skin barrier function. Immunofluorescence staining for filaggrin (green), nuclei stained with DAPI (blue) and results combined (Merge). Magnification ×600, scale bar 50 μm. Negative control (NC); infected with S. pyogenes (IN); vaseline treatment as placebo after infection (Pla); S. pyogenes infection with 1, 100, and 500 μg/mL BV cream (BV1, BV100, and BV500); S. pyogenes infection with 1, 100, and 500 μg/mL Mel cream (Mel1, Mel100, and Mel500).